本文選題：大環(huán)肽 + 包涵體　； 參考：《廣東藥科大學》2017年碩士論文

【摘要】：本課題主要內(nèi)容為大環(huán)肽241b的重組表達、純化工藝與生物功能的研究。涉及對目的大環(huán)肽241b的表達與富集、包涵體處理條件與大環(huán)肽純化過程優(yōu)化;采用抑菌試驗、細胞實驗和秀麗隱桿線蟲等不同方法檢測目的大環(huán)肽具有的生物學功能;對大環(huán)肽發(fā)酵過程中質(zhì)量控制及對污染菌株進行鑒定,并基于秀麗隱桿線蟲檢測其對241b生物學活性的影響。通過對上述幾方面內(nèi)容研究,獲得如下結(jié)果:1、在實驗室前期表達優(yōu)化后的基礎上,獲得可溶性目的大環(huán)肽241b。并對沉淀包涵體中目的大環(huán)肽的釋放條件進行優(yōu)化,獲得一組包涵體釋放的工藝條件,即:使用8 mol/L的尿素溶解包涵體后,獲得的上清液在含有1 mmol/L GSH,0.1 mmol/L GSSG,0.8 mmol/L L-Arg的透析液中,于25°C條件下進行梯度透析,最終每克包涵體可獲得約1.4 mg大環(huán)肽。2、采用C18固相萃取對可溶性蛋白進行脫鹽處理,70%ACN進行洗脫;脫鹽后大環(huán)肽經(jīng)程序為:0-5 min(88%A相:5%ACN+0.05%TFA,12%B相:90%ACN);5.01-10 min(40%A相,60%B相);10.01-30(10%A相,90%B相);30-30.10(88%A相,12%B相);30.10-40(88%A相,12%B相)的RP-HPLC進行洗脫;經(jīng)MOLDI-TOF-MS鑒定所得目的大環(huán)肽為241b。3、MIC法測定大環(huán)肽241b對G-菌,大腸桿菌、綠膿桿菌最小抑菌濃度分別為0.2、1.0 mg/m L;對G+菌枯草芽孢桿菌和金黃色葡萄球菌的最小抑菌濃度均為0.2mg/m L。CCK-8法檢測241b在低濃度時對MCF-7生長具有促進作用,高濃度具有抑制作用:C241b=0.03 mg/m L時,較對照組細胞活性增加38.44%;C241b=1mg/m L時達到最大抑制效果,達95%,IC50值為0.225 mg/m L。當241b與Cd Cl2共同作用于MCF-7時,展現(xiàn)出明顯的不規(guī)律性,說明兩者在適當?shù)臐舛葪l件下可發(fā)生絡合作用。對L1秀麗隱桿線蟲飼喂241b培養(yǎng)48 h,C241b=0.5 mg/m L時,對線蟲生長抑制效果達100%;濃度為1 mg/m L時,線蟲全部死亡。當C241b=0.05mg/m L時,線蟲中值壽命降低36.36%;隨著大環(huán)肽濃度的增加而逐漸減少;當C241b=2 mg/m L時,中值壽命降低90.9%;繼續(xù)增加大環(huán)肽濃度,已無明顯影響。該結(jié)果說明大環(huán)肽對線蟲的生長具有較強的抑制作用。4、對比相同條件下,搖瓶和攪拌式發(fā)酵罐發(fā)酵方式對大環(huán)肽的產(chǎn)量影響。優(yōu)化條件后,確定:241b菌液接種量為5%,加入葡萄糖終濃度為10 g/L LB中,培養(yǎng)至OD600≈1.2時,IPTG誘導表達8 h后,搖瓶與發(fā)酵罐中菌體量分別為3.5 g/L和5.38 g/L,菌體質(zhì)量提高了34.76%;包涵體中目的蛋白含量減少30.43%;最終搖瓶中每升發(fā)酵液可獲得44.91 mg大環(huán)肽241b,而發(fā)酵罐中可獲得68.57 mg,產(chǎn)量提高34.5%。說明在原有表達條件基礎上進一步擴大培養(yǎng),可提高大環(huán)肽產(chǎn)量。5、采用16S r DNA技術,鑒定污染的241b保存菌種241b-5和241b-7為粘質(zhì)沙雷氏菌的兩個亞型。基于秀麗隱桿線蟲模型,檢測其對241b生物學活性影響。發(fā)現(xiàn)單獨添加241b-5、241b-7時,均會降低線蟲的生長速率、壽命及身體擺動頻率;而與241b-1混合添加時,兩者對線蟲的生長速度均具有明顯影響,而對頭部擺動頻率影響較小;且241b-5與241b-1菌液混合時對線蟲壽命的影響與241b-1無明顯差別;當241b-7與241b-1混合時,線蟲壽命相比于241b-1單獨添加時降低了16%。為保證發(fā)酵質(zhì)量,應在實驗操作過程控制好實驗衛(wèi)生條件。綜上所述,本文通過重組表達技術獲得具有生物活性的大環(huán)肽,為重組大環(huán)肽的工業(yè)化生產(chǎn)提供了理論依據(jù),也為本實驗室后期突變體庫的構(gòu)建奠定了基礎。
[Abstract]:The main content of this project is the recombinant expression of macrocyclic peptide 241b, the purification process and the biological function research. It involves the expression and enrichment of the target macrocyclic peptide 241b, the conditions of inclusion body treatment and the optimization of the purification process of macrocyclic peptide, and the biological work of the macrocyclic peptide detection by the different methods of bacteriostasis test, cell experiment and Caenorhabditis elegans. Quality control and identification of contaminated strains in the fermentation process of macrocyclic peptide and the detection of the effect of Caenorhabditis elegans on the biological activity of 241b. The following results were obtained by studying the contents of these aspects. 1, on the basis of the optimization of the early expression of the laboratory, the soluble target macrocyclic peptide 241b. was obtained and the inclusion inclusion inclusion inclusion was obtained. The release conditions of the target macrocyclic peptide in the body were optimized, and a group of process conditions for the release of inclusion bodies were obtained, that is, after the inclusion body was dissolved with 8 mol/L urea, the obtained supernatant was in the dialysate containing 1 mmol/L GSH, 0.1 mmol/L GSSG, 0.8 mmol/L L-Arg, and at 25 degree C, the inclusion body could get about 1.4 m. G macrocyclic peptide.2 was desalted by C18 solid phase extraction and eluted by 70%ACN; after desalination, the macrocyclic peptide was programmed to be 0-5 min (88%A phase: 5%ACN+0.05%TFA, 12%B phase: 90%ACN); 5.01-10 min (40%A phase); The purpose of MOLDI-TOF-MS identification was 241b.3. The minimum inhibitory concentration of macrocyclic peptide 241b for G-, Escherichia coli and Pseudomonas aeruginosa was 0.2,1.0 mg/m L, and the minimum inhibitory concentration for Bacillus subtilis and Staphylococcus aureus were 0.2mg/m L.CCK-8 method, which promoted the growth of 241b at low concentration. Effect, high concentration has inhibitory effect: C241b=0.03 mg/m L, the cell activity of the control group increased by 38.44%, C241b=1mg/m L reached the maximum inhibitory effect, reached 95%, IC50 value was 0.225 mg/m L. when 241b and Cd Cl2 acted on MCF-7, showing obvious irregularity, suggesting that the two could have complex action under the appropriate concentration conditions. L1 Caenorhabditis elegans feeding 241b culture 48 h, C241b=0.5 mg/m L, the inhibitory effect on the growth of nematodes reached 100%; when the concentration was 1 mg/m L, the nematodes all died. When C241b=0.05mg/m L, the median life of nematodes decreased by 36.36%; as the concentration of macrocyclic peptides increased, the median life expectancy decreased by 90.9%; continue to increase large rings. The results showed that the peptide concentration had no obvious influence. The results showed that the macrocyclic peptide had a strong inhibitory effect on the growth of nematode.4. Under the same condition, the production of macrocyclic peptide was influenced by the fermentation mode of shake flask and stirred fermenting tank. The optimum conditions were determined: the inoculation amount of 241b bacteria was 5%, and the final concentration of glucose was 10 g/L LB, and it was cultured to OD600 1.2 When IPTG was induced to express 8 h, the amount of bacteria in the shake flask and the fermenting tank were 3.5 g/L and 5.38 g/L, respectively, the mass of the bacteria increased by 34.76%, the content of the target protein in the inclusion body decreased by 30.43%, and the final fermentation liquid in the shake flask could obtain 44.91 mg macrocyclic peptide 241b, and the fermentation tank could obtain 68.57 mg, and the yield increased 34.5%. in the original expression basis. On the basis of further expansion, the yield of macrocyclic peptide could be increased by.5, and 16S R DNA technology was used to identify the two subtypes of 241b-5 and 241b-7 contaminated 241b species, 241b-5 and 241b-7. Based on the Caenorhabditis elegans model, the effects on the biological activity of 241b were detected. It was found that the growth rate of the nematode could be reduced when 241b-5241b-7 was added alone. Life and the frequency of body wobble; when mixed with 241b-1, both of them have an obvious effect on the growth speed of the nematode, but less on the frequency of the head swing, and there is no significant difference in the effect of the 241b-5 and 241b-1 mixture on the life of the nematode when mixed with the 241b-1. When 241b-7 and 241b-1 are mixed, the life of the nematode is added to the 241b-1 alone. In order to reduce the quality of 16%. to ensure the quality of fermentation, we should control the experimental sanitary conditions in the process of experimental operation. In summary, the recombinant expression technology is used to obtain macrocyclic peptides with biological activity, which provides a theoretical basis for the industrial production of recombinant macrocyclic peptide and the foundation for the construction of the later mutant library of the laboratory.

【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R945

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